1,2,4-benzotriazine oxides as radiosensitizers and selective cytotoxic agents

ABSTRACT

A method of using 1,2,4-benzotriazine oxides, some of which are novel compounds, as radiosensitizers and selective cytotoxic agents is disclosed. These compounds are shown to specifically radiosensitize hypoxic tumor cells. Some are additionally disclosed to be useful as specific cytotoxic agents for these cells. They also show an unexpected ability to radiosensitize aerobic cells following or preceding a hypoxic incubation of the cells with the drug. This provides a basis for selective radiosensitization of tumors compared to normal cells. A novel method for preparing the 1,2,4-benzotriazine oxides is also disclosed.

REFERENCE TO GOVERNMENT GRANT OR CONTRACT

The invention described herein was made in the course of work undergrant or contract from the Department of Health and Human Services. TheGovernment has certain rights in this invention.

This application is a division of application Ser. No. 08/378,420, filedJan. 26, 1995, which in turn is a division of application Ser. No.07/939,787 filed Oct. 27, 1992, now abandoned, which is a division ofapplication Ser. No 07/409,480, filed Sep. 19, 1989, now U.S. Pat. No.5,475,287, which is a continuation-in-part of U.S. application Ser. No.356,602, filed 24 May 1989, now abandoned, which was a continuation ofU.S. application Ser. No. 169,873, filed 18 Mar. 1988, now abandoned,which in turn was a continuation-in-part of U.S. application Ser. No.911,906, filed 25 Sep. 1986, also abandoned.

TECHNICAL FIELD

The invention relates to cytotoxic agents and radiotherapy effectiveagainst hypoxic cells. More specifically, the invention relates tocertain novel 1,2,4-benzotriazine oxides, to methods of selectivelykilling tumor cells and/or sensitizing tumor cells to radiation usingselected 1,2,4-benzotriazine oxides, and to novel synthetic methods.

BACKGROUND ART

Hypoxic cell radiosensitizers are compounds that selectively increasethe sensitivity of hypoxic cells to destructive radiation. Cytotoxinswhich have enhanced activity under hypoxic conditions also provide ameans for selective destruction of cells under low oxygen pressure. Thisspecificity for hypoxic cells is important because it is tumors that aretypically characterized by such cells. Virtually all tumors which arepresent as solid masses contain these cells, while normal cellsgenerally have an adequate supply of oxygen. Accordingly, anti-tumoragents can be made selective for tumors by virtue of high activity underhypoxic conditions, and radiation can be employed more effectively inthe presence of these sensitizers.

Of course, the use of radiation treatment to destroy tumor cells is onlypractical if damage to the surrounding normal tissue can be minimized oravoided. The effects of radiation are enhanced by the presence ofoxygen, and it is established that as the dose of radiation isincreased, the effectiveness of the radiation in destroying target cellsis enhanced most dramatically when oxygen is present. Therefore,selectivity for tumor cells toward radiation is difficult toachieve--normal cells, in view of their oxygen supply, are generallymore susceptible to radiation than the target tumor cells. It istherefore desirable to provide a means of sensitizing tumor cells, butnot the surrounding tissue, to radiation treatment. One solution wouldbe to increase the supply of oxygen to these tumor cells. This, however,has proved difficult to do.

Various heterocyclic compounds and in particular those with oxidizednitrogen moieties, have been used to radiosensitize hypoxic tumor cells.Indeed, it has been postulated that the oxidized nitrogen functionalityis responsible for this activity. Nitroimidazoles, particularlymisonidazole (MIS) and metronidazole have been studied extensively, andMIS is commonly used as a standard in in vitro and in vivo tests forradiosensitizing activity. (See, e.g., Asquith, et al, Radiation Res(1974) 60:108-118; Hall, et al, Brit J Cancer (1978) 37: 567-569; Brown,et al, Radiation Res (1980) 82:171-190; and U.S. Pat. No. 4,371,540. Theradiosensitizing activities of certain 1-substituted3(5)-nitro-s-triazoles and of various quinoxaline-1,4-dioxidederivatives have also been disclosed.

In addition, U.S. Ser. Nos. 730,761, filed 3 May 1985, and 788,762,filed 18 Oct. 1985 assigned to the same assignee and incorporated byreference disclose a group of radiosensitizers that do not containoxidized nitrogen--the substituted benzamides and nicotinamides andtheir thio analogs. These compounds, nevertheless, are radiosensitizers.It is important to distinguish the ability to sensitize hypoxic cellsselectively, for instance, by enhancing their oxygen supply, fromanother mechanism commonly encountered for "sensitizing" cells:inhibition of the enzyme poly(ADP-ribose)polymerase, which is believedto be essential in the repair of irradiated cells after radiation. Thisrepair mechanism is operative in both hypoxic tumor cells and in normalcells. Hence, administration of "radiosensitizers" which operateaccording to this latter mechanism does not accomplish the desiredpurpose of selectively sensitizing the target tumor cells.

A group of compounds which has not previously been suggested for use ineither selectively killing hypoxic cells or in radiosensitizing suchcells is 3-amino-1,2,4-benzotriazine 1,4-di-N-oxide and relatedcompounds. Related U.S. Pat. Nos. 3,980,779; 3,868,371; and 4,001,410disclose the preparation of a group of these compounds and their use asanti-microbial agents, particularly by addition of these materials tolivestock fodder. U.S. Pat. Nos. 3,991,189 and 3,957,799 disclosederivatives of these compounds bearing substituents on the nitrogen ofthe 3-amino group. These compounds also have anti-microbial activity.

The present invention provides additional compounds which specificallyradiosensitize hypoxic cells and which, furthermore, are directlycytotoxic to hypoxic cells both in vitro and in vivo. Therefore,administration of these compounds prior to or following radiationtreatment of tumors selectively kills the hypoxic (tumor) cells whichsurvive the radiation dose. Both the ability of these compounds toradiosensitize hypoxic cells and especially their ability to selectivelykill hypoxic cells directly are unexpected properties of thesecompounds.

The invention also provides novel 1,2,4-benzotriazine oxides useful asradiosensitizers and/or selective cytotoxic agents; methods ofsynthesizing the compounds; and methods of administering the compoundsto achieve radiosensitization and/or selective cell killing.

DISCLOSURE OF THE INVENTION

The invention provides a valuable addition to the group of compoundscurrently available as selective radiosensitizers and selectivecytotoxic agents for hypoxic tumor cells. Some of the compounds nownewly shown to be useful in this regard are known compounds. Others arethemselves novel.

Accordingly, one aspect of the invention is a method of radiosensitizinghypoxic tumor cells by administering to these cells a compound of theformula: ##STR1## wherein X is H; hydrocarbyl (1-4C); hydrocarbyl (1-4C)substituted with OH, NH₂, NHR or NRR; halogen; OH; alkoxy (1-4C); NH₂ ;NHR or NRR; wherein the various R groups are independently selected fromlower alkyl (1-4C) and lower acyl (1-4C) and the R's may themselves besubstituted with OH, NH₂, alkyl (1-4C) secondary and dialkyl (1-4C)tertiary amino groups, alkoxy (1-4C) or halogen. In the case of NRR, thetwo R's can be linked together directly or through a bridge oxygen intoa morpholino ring, pyrrolidino ring or piperidino ring;

n is 0 or 1; and

Y¹ and Y² are independently either H; nitro; halogen; hydrocarbyl(1-14C) including cyclic and unsaturated hydrocarbyl, optionallysubstituted with 1 or 2 substituents selected from the group consistingof halogen, hydroxy, epoxy, alkoxy (1-4C), alkylthio (1-4C), primaryamino (NH₂), alkyl (1-4C) secondary amino, dialkyl (1-4C) tertiaryamino, dialkyl (1-4C) tertiary amino where the two alkyls are linkedtogether to produce a morpholino, pyrrolidino or piperidino, acyloxy(1-4C), acylamido (1-4C) and thio analogs thereof, acetylaminoalkyl(1-4C), carboxy, alkoxycarbonyl (1-4C), carbamyl, alkylcarbamyl (1-4C),alkylsulfonyl (1-4C) or alkylphosphonyl (1-4C), wherein the hydrocarbylcan optionally be interrupted by a single ether (--O--) linkage; orwherein Y¹ and Y² are independently either morpholino, pyrrolidino,piperidino, NH₂, NHR', NR'R' O(CO)R', NH(CO)R', O(SO)R', or O(POR')R' inwhich R' is a hydrocarbyl (1-4C) which may be substituted with OH, NH₂,alkyl (1-4C) secondary amino, dialkyl (1-4C) tertiary amino, morpholino,pyrrolidino, piperidino, alkoxy (1-4C), or halogen substituents, orpharmacologically acceptable salts of said compound.

In another aspect, the invention provides an improved method offractionated radiotherapy which involves treating the cells requiringradiotherapy with a 1,2,4-benzotriazine oxide of Formula (I), as justdefined, before or after subjecting the treated cells to a plurality ofdistinct radiation doses over an extended period of time, each of theradiation doses being less than about 5 Gy.

The compounds useful in conjunction with the presently disclosedradiosensitizing methods, therefore, are the mono- or dioxides ofoptionally substituted 1,2,4-benzotriazine which may contain ahydrocarbyl (1-4C), hydroxyl, alkoxy or amino group, either substitutedor unsubstituted, in the 3-position, and their pharmacologicallyacceptable salts as set forth in Formula I

The invention also provides a method for selectively killing hypoxictumor cells using certain of these 1,2,4-benzotriazine oxides. Thecompounds which are useful as selective cytotoxic agents are a subset ofthe above-defined compounds useful as radiosensitizers. That is, whileall of the compounds defined by Formula (I) are generally effective asradiosensitizers, only those compounds unsubstituted at the 3-positionor having a 3-amino or 3-hydrocarbyl (1-4C) substituent (i.e., X═H,hydrocarbyl (1-4C), NH₂, NHR or NRR with each R as defined above) andwhich are di-N-oxides (n=1) are effective cytotoxic agents. In thisaspect, the invention provides a method of selectively killing hypoxictumor cells by administering one or more of these compounds (or itssalts) to the hypoxic tumor cells.

Certain of the compounds encompassed by Formula (I) are already known inthe art as being useful for other purposes; other compounds are novel.The novel compounds encompassed by the present invention and which maybe prepared by methods disclosed herein include compounds represented byFormula (I), in which the substituents fall into the following threeclasses:

I. X is OH, alkoxy (1-4C), NHR or NRR where each R is independently analkyl of 1-4 carbon atoms, or acyl of 1-4 carbon atoms, or where the twoR groups are alkyls linked together to form a pyrrolidino or piperidinoring or linked through an oxygen to form a morpholino ring, and the Rgroups may be further substituted with OH, NH₂, alkyl (1-4C) secondaryamino, dialkyl (1-4C) tertiary amino, pyrrolidino, piperidino, alkoxy(1-4C), or halogen substituents;

n is 1; and

Y¹ and Y² are independently either H; nitro; halogen; hydrocarbyl(1-14C) including cyclic and unsaturated hydrocarbyl, optionallysubstituted with 1 or 2 substituents selected from the group consistingof halogen, hydroxy, epoxy, alkoxy (1-4C), alkylthio (1-4C), primaryamino (NH₂), alkyl (1-4C) secondary amino, dialkyl (1-4C) tertiaryamino, dialkyl tertiary amino where the two alkyls are linked togetherto produce a morpholino, pyrrolidino or piperidino, acyloxy (1-4C),acylamido (1-4C) and thio analogs thereof, acetylaminoalkyl (1-4C),carboxy, alkoxycarbonyl (1-4C), carbamyl, alkylcarbamyl (1-4C),alkylsulfonyl (1-4C) or alkylphosphonyl (1-4C), wherein the hydrocarbylcan optionally be interrupted by a single ether (--O--) linkage; orwherein Y¹ and Y² are independently either morpholino, pyrrolidino,piperidino, NH₂, NHR', NR'R' O(CO)R', NH(CO)R', O(SO)R', or O(POR')R' inwhich R' is a hydrocarbyl (1-4C) which may be substituted with OH, NH₂,alkyl (1-4C) secondary amino, dialkyl (1-4C) tertiary amino, morpholino,pyrrolidino, piperidino, alkoxy (1-4C), or halogen substituents.Pharmacologically acceptable salts of these compounds are also includedin this class of compounds.

II. X is NH_(2;)

n is 1; and

Y¹ and Y² are chosen such that one but not both may be hydrogen and oneor both may independently be either nitro, saturated or unsaturatedhydrocarbyl of 7-14C, or unsaturated hydrocarbyl of 2-6C, optionallysubstituted with 1 or 2 substituents selected from the group consistingof halogen, hydroxy, epoxy, alkoxy (1-4C), alkylthio (1-4C), primaryamino (NH₂), alkyl (1-4C) secondary amino, dialkyl (1-4C) tertiaryamino, dialkyl tertiary amino where the two alkyls are linked togetherto produce a morpholino, pyrrolidino or piperidino, acyloxy (1-4C),acylamido (1-4C) and thio analogs thereof, acetylaminoalkyl (1-4C),carboxy, alkoxycarbonyl (1-4C), carbamyl, alkylcarbamyl (1-4C),alkylsulfonyl (1-4C) and alkylphosphonyl (1-4C), wherein the hydrocarbylcan optionally be interrupted by a single ether (--O--) linkage; orwherein Y¹ and Y² are independently either morpholino, pyrrolidino,piperidino, NH₂, NHR', NR'R' O(CO)R', NH(CO)R', O(SO)R', or O(POR')R' inwhich R' is a hydrocarbyl (1-4C) which may be substituted with OH, NH₂,alkyl (1-4C) secondary amino, dialkyl (1-4C) tertiary amino, morpholino,pyrrolidino, piperidino, alkoxy (1-4C), or halogen substituents.Pharmacologically acceptable salts of these compounds are also includedin this class of compounds.

III. X is hydrogen or hydrocarbyl (2-4C) optionally substituted with OH,NH₂, alkoxy (1-4C) or halogen substituents;

n is 1; and

y¹ and Y² are independently either H; nitro; halogen; hydrocarbyl(1-14C) including cyclic and unsaturated hydrocarbyl, optionallysubstituted with 1 or 2 substituents selected from the group consistingof halogen, hydroxy, epoxy, alkoxy (1-4C), alkylthio (1-4C), primaryamino (NH₂), alkyl (1-4C) secondary amino, dialkyl (1-4C) tertiaryamino, dialkyl tertiary amino where the two alkyls are linked togetherto produce a morpholino, pyrrolidino or piperidino, acyloxy (1-4C),acylamido (1-4C) and thio analogs thereof, acetylaminoalkyl (1-4C),carboxy, alkoxycarbonyl (1-4C), carbamyl, alkylcarbamyl (1-4C),alkylsulfonyl (1-4C) or alkylphosphonyl (1-4C), wherein the hydrocarbylcan optionally be interrupted by a single ether (--O--) linkage; orwherein Y¹ and Y² are independently either morpholino, pyrrolidino,piperidino, NH₂, NHR', NR'R' O(CO)R', NH(CO)R', O(SO)R', or O(POR')R' inwhich R' is a hydrocarbyl (1-4C) which may be substituted with OH, NH₂,alkyl (1-4C) secondary amino, dialkyl (1-4C) tertiary amino, morpholino,pyrrolidino, piperidino, alkoxy (1-4C), or halogen substituents.Pharmacologically acceptable salts of these compounds are also includedin this class of compounds.

The invention also provides a straightforward, one-step synthesis forpreparing 1,2,4-benzotriazine oxides unsubstituted at the 3-position(i.e., the compounds of Formula (I) wherein X═H) by treating thecorresponding 3-amino-1,2,4-benzotriazine oxide with a lower alkylnitrite under reductive deaminating conditions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B and 1C show the selective cytotoxicity of3-amino-1,2,4-benzotriazine 1,4-dioxide for hypoxic cells derived fromhamster, mouse and human tissues.

FIG. 2 shows the in vivo efficacy of 3-amino-1,2,4-benzotriazine1,4-dioxide in enhancing the killing of tumor cells when combined withradiation.

FIG. 3 shows the killing of tumor cells in vivo by3-amino-1,2,4-benzotriazine 1,4-dioxide when the tumor has been madehypoxic by the intraperitoneal administration of the antihypertensivedrug hydralazine.

FIG. 4 is a graph illustrating aerobic radiosensitization of CHO cellsby hypoxic pretreatment or post-treatment with3-amino-1,2,4-benzotriazine 1,4-dioxide, as described in Example 22.

FIG. 5 illustrates tumor cell survival of SCCVII tumor irradiated with8×2.5 Gy in four days, also as described in Example 22.

FIG. 6 graphically illustrates the growth delay of SCCVII tumorsirradiated with 8×2.5 Gy in four days, also as described in Example 22.

FIG. 7 shows in graph form the average skin reaction of normal mouseskin irradiated with 8 fractions (every 12 hours) of between 3 and 6 Gyper fraction.

MODES OF CARRYING OUT THE INVENTION

A. The Compounds Useful in the Invention

The compounds useful as radiosensitizers and selective cytotoxic agentsas described herein are derivatives of 1,2,4-benzotriazine oxiderepresented by Formula (I).

These compounds, as shown in the Formula (I), contain a group X in their3 position. X will vary specifically as set forth above, depending uponthe activity desired. Subject to the above-recited selection criteria, Xis chosen in general from hydrogen; unsubstituted hydrocarbyls (1-4C)such as methyl, ethyl, s-butyl and the like; hydroxy; alkoxy (1-4C) suchas methoxy, ethoxy, propoxy, t-butoxy and the like; primary amino (HH₂);secondary amino (NHR) where R is an alkyl or acyl of 1 to 4 carbons,such as methylamino, ethylamino and the like; tertiary amino (NRR) whereeach of the R groups is an alkyl or acyl of 1 to 4 carbons, for examplediethylamino and the like, or the two R's join to form a morpholino,pyrrolidino or piperidino ring. In the case of the various alkyl andacyl R groups, they can be further substituted with OH, NH₂, lower alkyl(1-4C) secondary amino and dialkyl (1-4C) tertiary amino, morpholinopyrrolidino, piperidino, alkoxy (1-4C) or halogen (fluoro, chloro, bromoor iodo) substituents.

In the case of the hydrocarbyl X groups, they can be further substitutedwith OH, NH₂, alkyl secondary amino, dialkyl tertiary amino, alkoxy(1-4C) or halogen (fluoro, chloro, bromo or iodo) substituents.

The compounds of Formula (I) additionally contain groups Y¹ and Y².These groups are selected specifically according to the criteria setforth above, depending upon the utility desired.

Subject to these criteria, Y¹ and Y² may be selected from hydrogen;nitro; halogen (e.g. fluoro, chloro, bromo or iodo); or hydrocarbyl(1-14C). When hydrocarbyl, Y¹ or Y² may be saturated or unsaturated,cyclic or acyclic, and may optionally be interrupted by a single etherlinkage. Thus, the unsubstituted hydrocarbyl forms of Y¹ or Y² can be,for example, methyl, ethyl, n-propyl, s-butyl, n-hexyl,2-methyl-n-pentyl, 2-ethoxyethyl, 3-(n-propoxy)-n-propyl,4-methoxybutyl, cyclohexyl, tetrahydrofurfuryl, furfuryl, cyclohexenyl,3-(n-decyloxy)-n-propyl, 4-methyloctyl, 4,7-dimethyloctyl, or the like.

The hydrocarbyl Y¹ and Y² groups may optionally be substituted with 1 or2 substituents selected from halogen such as fluoro, chloro, bromo oriodo; hydroxy; epoxy; alkoxy (1-4C) such as, for example, methoxy,n-propoxy and t-butoxy; alkyl thio; (1-4C) primary amino (NH₂);morpholino; pyrrolidino; piperidino; secondary amino (NHR') where R' isa 1-4C alkyl, such as methylamino, propylamino and the like; tertiaryamino (NR'R'); acyloxy and acylamido groups represented by R'COO-- andR'CONH--, respectively, and their thiol analogs represented by R'CSO--and R'CSNH-- respectively; carboxy (--C(O)OH); alkoxycarbonyl(--C(O)OR'); carbamyl (--C(O)NH₂); alkylcarbamyl (1-4C) (--C(O)NH');alkylsulfonyl (1-4C) (R'SO₂ --); and alkyl phosphonyl (1-4C)(R'P(OR')O--).

In addition Y¹ and Y² can each independently be --NH₂, --NHR', --NR'R',--OCOR', --NH(CO)R', 'O(SO)R-- or --O(POR')R' in which the various R'groups are lower alkyls (1-4C) which themselves may be substituted withOH, NH₂, alkyl secondary and tertiary amino, pyrrolidino, piperidino,alkoxy (1-4C), or halogen substituents.

A particularly promising class of compounds for use both asradiosensitizers and selective cytotoxic agents include thoserepresented by the following structural Formula (II): ##STR2## InFormula (II), one of Y¹ and Y² is H, the other an electron-withdrawinggroup (e.g., nitro, carboxy, alkoxycarbonyl, alkylsulfonyl), R₁ and R₂are independently selected from the group consisting of hydrogen andlower alkyl, or the R₁ and R₂ groups may be linked to form a piperidinoor pyrrolidino ring, and m is an integer from 0-4 inclusive, preferably1 or 2.

Where X is OH, of course, the compounds may also be prepared and used asthe pharmaceutically acceptable salts formed from inorganic bases, suchas sodium, potassium, or calcium hydroxide, or from organic bases, suchas caffeine, ethylamine, and lysine.

When X is NH₂, NHR, or NRR, e.g., NH--CH₂ --(CH₂)_(m) --CH₂ NR₁ R₂ as inFormula (II), pharmaceutically acceptable acid addition salts may beused. These salts are those with inorganic acids such as hydrochloric,hydrobromic or phosphoric acids or organic acids such as acetic acid,pyruvic acid, succinic acid, mandelic acid, p-toluene sulfonic acid, andso forth. (Amino substituents on the hydrocarbyl side chain can also, ofcourse, be converted to salts.)

The 1,2,4-benzotriazines may be used in the practice of this inventionas the mono- or dioxides; i.e. either the 1-nitrogen of the triazinoring may be oxidized, or both the 1-and 4-nitrogens may be oxidized.

Specific particularly preferred compounds which are useful in theradiosensitization and cytotoxic procedures of the invention include

3-hydroxy-1,2,4-benzotriazine 1-oxide;

3-hydroxy-1,2,4-benzotriazine 1,4-dioxide;

3-amino-1,2,4-benzotriazine 1-oxide;

3-amino-1,2,4-benzotriazine 1,4-di-oxide;

6(7)-methoxy-3-hydroxy-1,2,4-benzotriazine 1-oxide;

6(7)-methoxy-3-hydroxy-1,2,4-benzotriazine 1,4-dioxide;

6(7)-methoxy-3-amino-1,2,4-benzotriazine 1-oxide;

6(7)-methoxy-3-amino-1,2,4-benzotriazine 1,4-dioxide;

6(7)-ethoxy-3-hydroxy-1,2,4-benzotriazine 1-oxide;

6(7)-ethoxy-3-hydroxy-1,2,4-benzotriazine 1,4-dioxide;

6(7)-ethoxy-3-amino-1,2,4-benzotriazine 1-oxide;

6(7)-ethoxy-3-amino-1,2,4-benzotriazine 1,4-dioxide;

6(7)- 4-acetamido-n-butanoxy!-3-hydroxy-1,2,4-benzotriazine 1-oxide;

6(7)- 4-acetamido-n-butanoxy!-3-hydroxy-1,2,4-benzotriazine 1,4-dioxide;

6(7)- 4-acetamido-n-butanoxy!-3-amino-1,2,4-benzotriazine 1-oxide;

6(7)- 4-acetamido-n-butanoxy!-3-amino-1,2,4-benzotriazine 1,4-dioxide;

6(7)- 1-(2,3-dihydroxy)propoxy!-3-hydroxy-1,2,4-benzotriazine 1-oxide;

6(7)- 1-(2,3-dihydroxy)propoxy!-3-hydroxy-1,2,4-benzotriazine1,4-dioxide;

6(7)- 1-(2,3-dihydroxy)propoxy!-3-amino-1,2,4-benzotriazine 1-oxide;

6(7)- 1-(2,3-dihydroxy)propoxy!-3-amino-1,2,4-benzotriazine 1,4-dioxide;

6(7)- (2-furyl)methylamino!-3-hydroxy-1,2,4-benzotriazine 1-oxide;

6(7)- (2-furyl)methylamino!-3-hydroxy-1,2,4-benzotriazine 1,4-dioxide;

6(7)- (2-furyl)methylamino!-3-amino-1,2,4-benzotriazine 1-oxide;

6(7)- (2-furyl)methylamino!-3-amino-1,2,4-benzotriazine 1,4-dioxide;

6(7)-(2-methoxyethylamino)-3-hydroxy-1,2,4-benzotriazine 1-oxide;

6(7)-(2-methoxyethylamino)-3-hydroxy-1,2,4-benzotriazine 1,4-dioxide;

6(7)-(2-methoxyethylamino)-3-amino-1,2,4-benzotriazine 1-oxide;

6(7)-(2-methoxyethylamino)-3-amino-1,2,4-benzotriazine 1,4-dioxide;

6(7)-carbethoxymethoxy-3-hydroxy-1,2,4-benzotriazine 1-oxide;

6(7)-carbethoxymethoxy-3-hydroxy-1,2,4-benzotriazine 1,4-dioxide;

6(7)-carbethoxymethoxy-3-amino-1,2,4-benzotriazine 1-oxide;

6(7)-carbethoxymethoxy-3-amino-1,2,4-benzotriazine 1,4-dioxide;

6(7)- (2-methoxyethyl)carbamylmethoxy!-3-hydroxy-1,2,4-benzotriazine1-oxide;

6(7)- (2-methoxyethyl)carbamylmethoxy!-3-hydroxy-1,2,4-benzotriazine1,4-dioxide;

6(7)- (2-methoxyethyl)carbamylmethoxy!-3-amino-1,2,4-benzotriazine1-oxide;

6(7)- (2-methoxyethyl)carbamylmethoxy!-3-amino-1,2,4-benzotriazine1,4-dioxide;

6(7)- (2-hydroxyethyl)carbamylmethoxy!-3-hydroxy-1,2,4-benzotriazine1-oxide;

6(7)- (2-hydroxyethyl)carbamylmethoxy!-3-hydroxy-1,2,4-benzotriazine1,4-dioxide;

6(7)- (2-hydroxyethyl)carbamylmethoxy!-3-amino-1,2,4-benzotriazine1-oxide;

6(7)- (2-hydroxyethyl)carbamylmethoxy!-3-amino-1,2,4-benzotriazine1,4-dioxide;

6(7)- 1-(2-hydroxy-3-morpholino)propoxy!-3-hydroxy-1,2,4-benzotriazine1-oxide;

6(7)- 1-(2-hydroxy-3-morpholino)propoxy!-3-hydroxy-1,2,4-benzotriazine1,4-dioxide;

6(7)- 1-(2-hydroxy-3-morpholino)propoxy!-3-amino-1,2,4 benzotriazine1-oxide;

6(7)- 1-(2-hydroxy-3-morpholino)propoxy!-3-amino-1,2,4 benzotriazine1,4-dioxide;

6(7)- 3-amino-n-propoxy!-3-hydroxy-1,2,4-benzotriazine 1-oxide;

6(7)- 3-amino-n-propoxy!-3-hydroxy-1,2,4-benzotriazine 1,4-dioxide;

6(7)- 3-amino-n-propoxy!-3-amino-1,2,4-benzotriazine 1-oxide;

6(7)- 3-amino-n-propoxy!-3-amino-1,2,4-benzotriazine 1,4-dioxide;

6(7)- 2,3-epoxypropoxy!-3-hydroxy-1,2,4-benzotriazine 1-oxide;

6(7)- 2,3-epoxypropoxy!-3-hydroxy-1,2,4-benzotriazine 1,4-dioxide;

6(7)- 2,3-epoxypropoxy!-3-amino-1,2,4-benzotriazine 1-oxide;

6(7)- 2,3-epoxypropoxy!-3-amino-1,2,4-benzotriazine 1,4-dioxide;

6(7)- 3-methoxy-2-hydroxy-n-propoxy!-3-hydroxy-1,2,4-benzotriazine1-oxide;

6(7)- 3-methoxy-2-hydroxy-n-propoxy!-3-hydroxy-1,2,4-benzotriazine1,4-dioxide;

6(7)- 3-methoxy-2-hydroxy-n-propoxy!-3-amino-1,2,4-benzotriazine1-oxide;

6(7)- 3-methoxy-2-hydroxy-n-propoxy!-3-amino-1,2,4-benzotriazine1,4-dioxide;

6(7)- 4-ethoxy-3-hydroxy-n-butoxy!-3-hydroxy-1,2,4-benzotriazine1-oxide;

6(7)- 4-ethoxy-3-hydroxy-n-butoxy!-3-hydroxy-1,2,4-benzotriazine1,4-dioxide;

6(7)- 4-ethoxy-3-hydroxy-n-butoxy!-3-amino-1,2,4-benzotriazine 1-oxide;

6(7)- 4-ethoxy-3-hydroxy-n-butoxy!-3-amino-1,2,4-benzotriazine1,4-dioxide;

6(7)- 3,4-dihydroxy-n-butoxy!-3-hydroxy-1,2,4-benzotriazine 1-oxide;

6(7)- 3,4-dihydroxy-n-butoxy!-3-hydroxy-1,2,4-benzotriazine 1,4-dioxide;

6(7)- 3,4-dihydroxy-n-butoxy!-3-amino-1,2,4-benzotriazine 1-oxide;

6(7)- 3,4-dihydroxy-n-butoxy!-3-amino-1,2,4-benzotriazine 1,4-dioxide;

6(7)-methyl-3-hydroxy-1,2,4-benzotriazine 1-oxide;

6(7)-methyl-3-hydroxy-1,2,4-benzotriazine 1,4-dioxide;

6(7)-methyl-3-amino-1,2,4-benzotriazine 1-oxide;

6(7)-methyl-3-amino-1,2,4-benzotriazine 1,4-dioxide;

6(7)-ethyl-3-hydroxy-1,2,4-benzotriazine 1-oxide;

6(7)-ethyl-3-hydroxy-1,2,4-benzotriazine 1,4-dioxide;

6(7)-ethyl-3-amino-1,2,4-benzotriazine 1-oxide;

6(7)-ethyl-3-amino-1,2,4-benzotriazine 1,4-dioxide;

6(7)-chloroacetamido-3-hydroxy-1,2,4-benzotriazine 1-oxide;

6(7)-chloroacetamido-3-hydroxy-1,2,4-benzotriazine 1,4-dioxide;

6(7)-chloroacetamido-3-amino-1,2,4-benzotriazine 1-oxide;

6(7)-chloroacetamido-3-amino-1,2,4-benzotriazine 1,4-dioxide;

6(7)- (2-hydroxyethyloxy)acetamido!-3-hydroxy-1,2,4-benzotriazine1-oxide;

6(7)- (2-hydroxyethyloxy)acetamido!-3-hydroxy-1,2,4-benzotriazine1,4-dioxide;

6(7)- (2-hydroxyethyloxy)acetamido!-3-amino-1,2,4-benzotriazine 1-oxide;

6(7)- (2-hydroxyethyloxy)acetamido!-3-amino-1,2,4-benzotriazine1,4-dioxide;

6,7-dimethoxy-3-hydroxy-1,2,4-benzotriazine 1-oxide;

6,7-dimethoxy-3-hydroxy-1,2,4-benzotriazine 1,4-dioxide;

6,7-dimethoxy-3-amino-1,2,4-benzotriazine 1-oxide;

6,7-dimethoxy-3-amino-1,2,4-benzotriazine 1,4-dioxide;

6,7-diethoxy-3-hydroxy-1,2,4-benzotriazine 1-oxide;

6,7-diethoxy-3-hydroxy-1,2,4-benzotriazine 1,4-dioxide;

6,7-diethoxy-3-amino-1,2,4-benzotriazine 1-oxide;

6,7-diethoxy-3-amino-1,2,4-benzotriazine 1,4-dioxide;

6(7)-propionyl-3-hydroxy-1,2,4-benzotriazine 1-oxide;

6(7)-propionyl-3-hydroxy-1,2,4-benzotriazine 1,4-dioxide;

6(7)-propionyl-3-amino-1,2,4-benzotriazine 1-oxide;

6(7)-propionyl-3-amino-1,2,4-benzotriazine 1,4-dioxide;

6(7)-(2-acetoxyethoxy)-3-hydroxy-1,2,4-benzotriazine 1-oxide;

6(7)-(2-acetoxyethoxy)-3-hydroxy-1,2,4-benzotriazine 1,4-dioxide;

6(7)-(2-acetoxyethoxy)-3-amino-1,2,4-benzotriazine 1-oxide;

6(7)-(2-acetoxyethoxy)-3-amino-1,2,4-benzotriazine 1,4-dioxide;

6(7)-n-hexyloxy-3-hydroxy-1,2,4-benzotriazine 1-oxide;

6(7)-n-hexyloxy-3-hydroxy-1,2,4-benzotriazine 1,4-dioxide;

6(7)-n-hexyloxy-3-amino-1,2,4-benzotriazine 1-oxide;

6(7)-n-hexyloxy-3-amino-1,2,4-benzotriazine 1,4-dioxide;

6(7)-ethylamino-3-hydroxy-1,2,4-benzotriazine 1-oxide;

6(7)-ethylamino-3-hydroxy-1,2,4-benzotriazine 1,4-dioxide;

6(7)-ethylamino-3-amino-1,2,4-benzotriazine 1-oxide;

6(7)-ethylamino-3-amino-1,2,4-benzotriazine 1,4-dioxide;

6(7)-(2-methoxyethoxy)-3-hydroxy-1,2,4-benzotriazine 1-oxide;

6(7)-(2-methoxyethoxy)-3-hydroxy-1,2,4-benzotriazine 1,4-dioxide;

6(7)-(2-methoxyethoxy)-3-amino-1,2,4-benzotriazine 1-oxide;

6(7)-(2-methoxyethoxy)-3-amino-1,2,4-benzotriazine 1,4-dioxide;

6(7)-(aminoacetamido)-3-hydroxy-1,2,4-benzotriazine 1-oxide;

6(7)-(aminoacetamido)-3-hydroxy-1,2,4-benzotriazine 1,4-dioxide;

6(7)-(aminoacetamido)-3-amino-1,2,4-benzotriazine 1-oxide;

6(7)-(aminoacetamido)-3-amino-1,2,4-benzotriazine 1,4-dioxide;

6(7)-(carbamylmethoxy)-3-hydroxy-1,2,4-benzotriazine 1-oxide;

6(7)-(carbamylmethoxy)-3-hydroxy-1,2,4-benzotriazine 1,4-dioxide;

6(7)-(carbamylmethoxy)-3-amino-1,2,4-benzotriazine 1-oxide;

6(7)-(carbamylmethoxy)-3-amino-1,2,4-benzotriazine 1,4-dioxide;

6(7)-(carboxymethoxy)-3-hydroxy-1,2,4-benzotriazine 1-oxide;

6(7)-(carboxymethoxy)-3-hydroxy-1,2,4-benzotriazine 1,4-dioxide;

6(7)-(carboxymethoxy)-3-amino-1,2,4-benzotriazine 1-oxide;

6(7)-(carboxymethoxy)-3-amino-1,2,4-benzotriazine 1,4-dioxide;

6(7)- 1,2-dihydroxyethyl!-3-amino-1,2,4-benzotriazine 1,4-dioxide;

6(7)- 1-(3-ethylamino-2-hydroxypropoxy)!-3-amino-1,2,4-benzotriazine1,4-dioxide;

6(7)- 2-ethylamino-1-hydroxyethyl!-3-amino-1,2,4-benzotriazine1,4-dioxide;

6(7)- 2-hydroxyethyl!-3-amino-1,2,4-benzotriazine 1,4-dioxide;

6(7)- 1-hydroxyethyl!-3-amino-1,2,4-benzotriazine 1,4-dioxide;

3-(2-hydroxyethylamino)-1,2,4-benzotriazine 1-oxide;

3-(2-hydroxyethylamino)-1,2,4-benzotriazine 1,4-dioxide;

6(7)-chloro-3-(2-hydroxyethylamino)-1,2,4-benzotriazine 1-oxide;

6(7)-chloro-3-(2-hydroxyethylamino)-1,2,4-benzotriazine 1,4-dioxide;

3-(1-hydroxyethylamino)-1,2,4-benzotriazine 1-oxide;

3-(1-hydroxyethylamino)-1,2,4-benzotriazine 1,4-dioxide;

1,2,4-benzotriazine 1-oxide;

1,2,4-benzotriazine 1,4-dioxide;

3-methyl-1,2,4-benzotriazine 1,4-dioxide;

3-ethyl-1,2,4-benzotriazine 1,4-dioxide;

3-propyl-1,2,4-benzotriazine 1,4-dioxide;

6(7)-amino-1,2,4-benzotriazine 1,4-dioxide;

6(7)-amino-3-methyl-1,2,4-benzotriazine 1,4-dioxide;

6(7)-amino-3-ethyl-1,2,4-benzotriazine 1,4-dioxide;

6(7)-methoxy-1,2,4-benzotriazine 1,4-dioxide;

6(7)-methoxy-3-methyl-1,2,4-benzotriazine 1,4-dioxide;

6(7)- 1-(2,3-dihydroxypropoxy!-1,2,4-benzotriazine 1,4-dioxide;

6(7)- 1,2-dihydroxyethyl!-1,2,4-benzotriazine 1,4-dioxide;

6(7)- 1-(3-ethylamino-2-hydroxypropoxy)!-1,2,4-benzotriazine1,4-dioxide;

6(7)- 2-ethylamino-1-hydroxyethyl!-1,2,4-benzotriazine 1-4 dioxide;

6(7)-chloro-1,2,4-benzotriazine 1,4-dioxide;

6(7)- 2-hydroxyethyl!-1,2,4-benzotriazine 1,4-dioxide;

6(7)- 1-hydroxyethyl!-1,2,4-benzotriazine 1,4-dioxide;

6(7)-nitro-3-amino-1,2,4-benzotriazine 1,4-dioxide;

3-(3-N,N-diethylaminopropylamino)-1,2,4-benzotriazine 1,4-dioxide;

6(7)-nitro-3-(2-N,N-diethylaminoethylamino)-1,2,4-benzotriazine1,4-dioxide

and their pharmaceutically acceptable salts and the thioamide analogs ofthe foregoing list of compounds. It should be noted that the Y¹ or Y²substituents set forth in most of the above compounds as present ineither the 6 or 7 positions (designated "6(7)") or in both the 6 and 7positions (designated "6,7") may also be present at the 5 and/or 8 ringpositions.

B. Preparation of the Compounds of the Invention

General methods for preparing some 3-amino derivatives are found in theabove-referenced patents to Ley et al., for example U.S. Pat No.3,980,779. The compounds are prepared from benzofuroxan of the formula:##STR3## by reaction with a salt of cyanamide, followed by acidificationof the reaction mixture. The benzofuroxan starting material is notsymmetric with respect to its own 5 and 6 positions (which are the 6 and7 positions of the resulting 3-amino benzotriazine oxide). Therefore, amixture of the 6- and 7-substituted materials may result. If desired,this mixture can be separated using conventional means into individualcomponents having a substituent in either the 6 or 7 position.

The dioxide may also be prepared from the parent monoxide or1,2,4-benzotriazine by peracid oxidation (see Robbins et al, J Chem Soc3186 (1957) and Mason et al, J Chem Soc B 911 (1970)).

In addition, the monoxide may be prepared by:

(1) cyclization of a 1-nitro-2aminobenzene compound using H₂ NCN.2HCl;

(2) oxidation of the parent compound given by the structure ##STR4## orby controlled reduction of the corresponding dioxide (see Mason, supra,and-Wolf et al, J Am Chem Soc 76:355 (1954)).

The 1,2,4-benzotriazines may be prepared by cyclization of formazanprecursors using BF₃ /AcOH (see Scheme I and Atallah and Nazer,Tetrahedron 38:1793 (1982)).

3-amino-1,2,4-benzotriazines may be prepared either by cyclization of aparent compound (see Scheme II and Arndt, Chem. Ber. 3522 (1913)) or byreduction of the monoxide or dioxide as above.

The 3-hydroxy-1,2,4-benzotriazine oxides may be prepared using peroxideand sodium tungstate (Scheme III), a novel synthetic procedure formaking the 3-hydroxy-1,4-dioxide compound, or concentrated sulfuric acidand sodium nitrate (Scheme IV). ##STR5##

The invention also encompasses a novel method of preparing1,2,4-benzotriazine oxides unsubstituted at the 3 position (sometimesreferred to herein as the "3-desamino" compounds). The novel synthesisinvolves reductive deamination of the corresponding 3-amino structure.In contrast to prior methods of synthesizing3-desamino-1,2,4-benzotriazine oxides, the present method enables asimple, straightforward one-step method which gives the desired productin a high yield. The method involves treating a 1,2,4-benzotriazineoxide of Formula (I), wherein X is NH₂, with a lower alkyl nitrite underreductive deaminating conditions. By "reductive deaminating conditions"is meant reaction conditions which will give rise to at least about 10%,preferably at least about 50%, of the desired 3-unsubstituted reactionproduct. A preferred lower alkyl nitrite for use in said method ist-butyl nitrite. Exemplary reductive deaminating conditions involvereaction in a compatible solvent, e.g., dimethyl formamide, at atemperature of at least about 60° C., typically at a temperature in therange of 60°-65° C. This reaction is illustrated generally at Scheme V,and is exemplified in Examples 12-15 herein. ##STR6## C. Formulation andAdministration

As demonstrated below, the oxidized benzotriazines of the invention maybe used to radiosensitize or selectively kill hypoxic tumor cells inwarm-blooded animal hosts. A way in which they may be used is inconjunction with agents known to selectively create hypoxia in tumors.Such methods include the use of antihypertensive drugs such ashydralazine, or agents which affect the amount of oxygen carried-by theblood. While these compounds will typically be used in cancer therapy ofhuman patients, they may be used to kill hypoxic tumor cells in otherwarm blooded animal species such as other primates, farm animals such ascattle, and sports animals and pets such as horses, dogs, and cats.

Hypoxia is believed to be associated with all types of solid malignantneoplasms. The compounds of the invention may, therefore, be used toradiosensitize or to kill neoplastic epithelial cells, endothelialcells, connective tissue cells, bone cells, muscle cells, nerve cells,and brain cells. Examples of carcinomas and sarcomas include carcinomassuch as epithelial cell, acidic cell, alveolar cell, basal cell, basalsquamous cell, cervical, renal, liver, Hurthle, Lucke, mucinous andWalker, and sarcomas such as Abernathy's, alveolar soft part,angiolithic, botyroid, encephaloid, endometria stroma, Ewing'sfascicular, giant cell, lymphatic, Jensen's, juxtacortical osteogenic,Kaposi's, medullary, and synovial. Specific examples of tumors that havebeen sensitized with other radiosensitizers are reported in Adams, G.E., Cancer: A Comprehensive Treatise (F. Becker, Ed) vol 6, pp 181-223,Plenum, N.Y., 1977.

The compounds may be administered to patients orally or parenterally(intravenously, subcutaneously, intramuscularly, intraspinally,intraperitoneally, and the like). When administered parenterally thecompounds will normally be formulated in a unit dosage injectable form(solution, suspension, emulsion) with a pharmaceutically acceptablevehicle. Such vehicles are typically nontoxic and nontherapeutic.Examples of such vehicles are water, aqueous vehicles such as saline,Ringer's solution, dextrose solution, and Hank's solution and nonaqueousvehicles such as fixed oils (e.g., corn, cottonseed, peanut, andsesame), ethyl oleate, and isopropyl myristate. Sterile saline is apreferred vehicle and the compounds are sufficiently water soluble toprovide a solution for all foreseeable needs. The vehicle may containminor amounts of additives such as substances that enhance solubility,isotonicity, and chemical stability, e.g., antioxidants, buffers, andpreservatives. When administered orally (or rectally) the compounds willusually be formulated into a unit dosage form such as a tablet, capsule,suppository or cachet. Such formulations typically include a solid,semisolid or liquid carrier or diluent. Exemplary diluents and vehiclesare lactose, dextrose, sucrose, sorbitol, mannitol, starches, gumacacia, calcium phosphate, mineral oil, cocoa butter, oil of theobroma,alginates, tragacanth, gelatin, methylcellulose, polyoxyethylenesorbitan monolaurate, methyl hydroxybenzoate, propyl hydroxybenzoate,talc, and magnesium stearate.

The amount of compound administered to the subject is sufficient toradiosensitize or to produce cytotoxicity in the malignant neoplasm tobe treated but below that which may elicit toxic effects to normaltissue. This amount will depend upon the type of tumor, the species ofthe subject being treated, the indicated dosage intended and the weightor body surface of the subject. The radiation may be administered tohumans in a variety of different fractionation regimes, i.e., the totalradiation dose is given in portions over a period of several days toseveral weeks. These are most likely to vary from daily (i.e., fivetimes per week) doses for up to six weeks, to once weekly doses for fourto six weeks. An individual dose of the benzotriazine will be givenbefore or after each radiation treatment and is likely to be in therange of 0.01 to 20 mmol/kg and usually in the range of 0.1 to 2mmol/kg. In these treatment regimens, each radiation dose is typically1-5 Gy, preferably, less than 2.5 Gy and more preferably 2-2.5Gy.Typically, one dose of radiation is administered per day although 2 ormore might be used if tolerated by the patient

It has now been found that the compounds disclosed herein asradiosensitizers, particularly 3-amino-1,2,4-benzotriazine 1,4-dioxide,both sensitize tumors to radiation without increasing the sensitivity ofnormal skin, and work in highly fractionated radiation regimens. Asdemonstrated in Example 22 herein, pre- or post-irradiation treatment ofcells with, for example, 3-amino-1,2,4-benzotriazine 1,4-dioxide, underhypoxic conditions, radiosensitizes cells even when the drug is notpresent during the radiation exposure and the cells are aerobic.

For use as selective cytotoxic agents, the compounds of the inventioncan be administered alone, with radiation or other cancer cytotoxicagents, with vasoactive drugs (e.g., hydralazine), or under otherconditions which reduce the amount of available oxygen carried by theblood such as anemia or drugs which increase the binding of oxygen tohemoglobin, all of which can enhance selectively the degree of hypoxiain the tumor.

EXAMPLES

The following examples further illustrate the compounds of the inventionand methods for synthesizing and using them, and are not intended tolimit the invention in any manner.

Experimental: All reactions were carried out in flame-dried glasswareand under a blanket of Argon. t-butyl nitrite (90%) was purchased fromthe Aldrich Chemical Company. Dimethylformamide was distilled fromcalcium hydride. 7-Nitro-3-amino-1,2,4-benzotriazine-3-amine 1-oxide waspurchased from Parish Chemical Company, trifluoroacetic anhydride,N,N-diethylethylene-diamine, N,N-diethylpropylenediamine and sodiumtungstate dihydrate were purchased from Aldrich Chemical Company and 70%hydrogen peroxide was a gift from Interox America. All reactants wereused without further purification. Flash chromatography was carried outon E. Merck 230-400 mesh silica gel under a positive pressure of argon.NMR spectra were obtained on a Varian XL-400 or Jeol FX90Q spectrometerand in d₆ -acetone, d₄ -methanol, or d₆ -dimethyl sulfoxide, asindicated, and are reported relative to the central peak in theappropriate multiplet (2.04, 3.30, and 2.49 ppm, respectively), UVspectra were obtained on a Perkin-Elmer 552 spectrophotometer in 95%ethanol, mass spectra were obtained on an LKB 9000 mass spectrometer,and elemental analyses were carried out by Desert Analytics, Tucson,Ariz.

Example 1

Preparation of 3-Hydroxy-1,2,4-Benzotriazine 1,4-Dioxide ##STR7##

A stirred mixture of 1.50 g (9.25 mmole) of 3-amino-1,2,4-benzotriazine1-oxide (1), 100.0 ml acetic acid, and 30.0 ml of 30% hydrogen peroxidewas treated with 3.05 g (9.25 mmole) of Na₂ WO₄ 2H₂ O. The mixture wasstirred in an oil bath at 60° C. for 4 days. The yellowish orangemixture was cooled to about 30° and filtered to remove a light yellownon-UV absorbing solid. The orange solution of hydrogen peroxide inacetic acid was evaporated to semi-dryness carefully with severaladditions of water and acetic acid to remove most of the peroxide. Theconcentrated solution was allowed to stand at room temperature to affordfour crops of an orange solid, 0.87 g (42% yield of the sodium salt of2). UV_(max) (20% CH₃ OH/H₂ O): 262.2 (ε39,460); 477 (ε7,030). IR(neat): 3530μ, 3150μ, 2650μ, 2180μ and 1635μ. Anal. (calculated for thesodium salt): C₇ H₄ N₃ O₃ Na 1.25H₂ O, 223.64: C,37.6; H,2.93; N,18.79.Found: C,37.8; H,2.75; N, 18.65. ##STR8##

Example 2

Preparation of 3-Amino-7-Trifluoromethyl-1,2,4-Benzotriazine 1-Oxide:##STR9##

A mixture of 4-chloro-3-nitrobenzotrifluoride (Aldrich, 2.70 g, 12.9mmole) and cyanamide dihydrochloride (2.75 g, 24 mmole) (previouslyprepared by treating an ether solution of cyanamide with HCl gas andcollecting the precipitated solid) was heated at 140° C. for 1 h. Theresidue was treated with 2N NaOH (45 ml), heated for a further 5 min,and then allowed to cool. The precipitate was collected, washed with H₂O, dried, and triturated with acetone-toluene to yield 1.32 g (45%) of 3as a light yellow solid M.P. 301°-302°, TLC: R_(f) 0.60 (9:1 methylenechloride:methanol on silica gel plates). Mass. Spec.: M⁺ =230 (q=100).

Example 3

Preparation of 3-Amino-7-Decyl-1,2,4-Benzotriazine 1-Oxide ##STR10##

Preparation of 4-(1-decyl)-2-nitroaniline: Acetic anhydride (400 ml) wasadded over a 30-minute period to a stirred solution of 4-decylaniline(Aldrich, 80 g, 0.34 mole) in hexanes (2.41). After stirring for 1 h,the mixture was cooled and treated over 30 min. at 5°-10° C. with 70%nitric acid (34 ml). Stirring was continued at 5°-10° C. for 1 h and at25° C. for 16 h. The mixture was diluted with H₂ O (11), stirred for 5h, poured into an open dish and allowed to stand for 16 h. After furtherdilution with H₂ O (1.51), the solid was collected and recrystallizedfrom an 85% ethanol solution (in water) to give 92 g (84%) of theintermediate as an orange solid, m.p. 64° C.

A solution (100 ml) of 85% KOH (19 g, 0.288 mole) in H₂ O was combinedwith a suspension of 4-(1-decyl)-2-nitroaniline (89 g, 0.28 mole),prepared above, in methanol (900 ml). The mixture was stirred for 6 h,neutralized to pH 7-8 with concentrated HCl, and evaporated in vacuo tonear dryness. After dilution with H₂ O (400 ml), the solid was collectedand air-dried to give 77 g (100%) of the intermediate as an orangesolid, m.p. 59° C.

1.0 g (8.7 mmole) of cyanamide dihydrochloride (previously prepared foruse by treating an ether solution of cyanamide with HCl gas andcollecting the precipitated solid) was added portionwise over 10 min toa preheated melt (190° C.) of 4-(1-decyl)-2-nitroaniline prepared in thepreceding step (500 mg, 1.8 mmole). The reaction mixture was heated at190° C. for 5 min, cooled to 25° C., treated with 6N KOH (10 ml), andheated at 90°-95° C. for 1 h. After cooling to 25° C., the solid wascollected, washed with H₂ O and ethanol and air-dried to give 0.25 g(46%) of compound 4 as a light yellow solid, m.p. 177° C. (dec). Mass.spec. M⁺ =285 (q=100), 302 (q=13).

Example 4

Preparation of 3-Amino-7-Carbamyl-1,2,4-Benzotriazine 1-Oxide ##STR11##

Preparation of 4-chloro-3-nitrobenzamide: 20.2 g (0.1 mole) of4-chloro-3-nitrobenzoic acid (Aldrich) and thionyl chloride (20 ml) werecombined, allowed to stand for 16 h, and refluxed for 4 h to give aclear red solution. The solution was evaporated in vacuo and azeotropedwith benzene. The residue was dissolved in acetonitrile (20 ml) andadded over 30 min to cold (-10° C.) concentrated ammonium hydroxide (100ml). After 3 h at -10° C. and 16 h at 25° C. the mixture was poured intoan open dish and allowed to evaporate to dryness. The residue wasslurried in H₂ O and the solid was collected and air-dried to give 19.8g (98%) of the intermediate as a light yellow solid, m.p. 153° C.

A solution of Na (3.45 g, 0.15 mole ) in ethanol (75 ml) was added to asolution of guanidine hydrochloride (15.8 g, 0.165 mole) in ethanol (75ml). After 1 h the mixture was filtered and the filtrate was combinedwith a suspension of 4-chloro-3-nitrobenzamide (10 g, 0.05 mole)prepared above, in ethanol (50 ml). The mixture was stirred and refluxedfor 16 h, cooled to 0°-5° C., and acidified with concentrated HCl (8ml). The collected solid was combined with K₂ CO₃ (28 g, 0.2 mole) andH₂ O (40 ml) and the mixture was stirred and heated at 100° C. for 8 h.After cooling to 25° C., the solid was collected, washed with H₂ O, andair-dried. The solid was suspended in boiling ethyl acetate, collectedand washed with hot ethyl acetate. The solid was repeatedly suspended inboiling dioxane and collected (6×100 ml). The combined filtrate wasevaporated in vacuo to a solid. The solid was suspended in 95% ethanol,collected and air-dried to give 0.44 g (4.3%) of compound 5 as a lightyellow solid, m.p. 300° C. TLC: R_(f) =0.23 (methylene chloride: acetoneof 2:1, silica gel plates). Mass. Spec.: M⁺ 205 (q=100).

Example 5

Preparation of 7-Acetyl-3-Amino-1,2,4-Benzotriazine 1-Oxide Oxime##STR12##

A combined mixture of 7-acetyl-3-amino-1,2,4-benzotriazine 1-oxide(prepared in Example 5; 50 mg, 0.25 mmole), hydroxylamine hydrochloride(200 mg, 2.88 mmole), pyridine (1 ml), and ethanol (1 ml) was heated at90°-95° C. for 1 h and then cooled to 25° C. The mixture was dilutedwith 95% ethanol (5 ml) and the solid was collected and air-dried togive 30 mg (56%) of compound 6 as a light yellow solid, m.p. 278° C.(dec). TLC: R_(f) =0.60 (9:1 methylene chloride:methanol). Mass Spec.:M⁺ =219 (q=100).

Example 6

Preparation of 3-Amino-6(7)-Decyl-1,2,4-Benzotriazine 1,4-Dioxide##STR13##

5-(1-decyl)-benzofuroxan: A combined mixture of4-(1-decyl)-2-nitroaniline (77 g, 0.28 mole), 5.25% NaOCl in H₂ O (476g, 0.34 mole), 85% KOH (20.3 g, 0.31 mole), nBu₄ NHSO₄ (4.7 g, 0.014mole), and CH₂ Cl₂ (2.28 l) was stirred rapidly for 6 h and diluted withH₂ O (500 ml) and CH₂ Cl₂ (1 l). The separated organic phase was washedsuccessively with 1N HCl (1 l) and brine (2×1 l)), dried (Na₂ SO₄), andconcentrated in vacuo to yield a red oil, 70 g (92%).

A solution of 5-(1-decyl)-benzofuroxan as prepared above (10 g, 0.036mole) and benzyltriethyl ammonium chloride (0.36 g, 0.0016 mole) in DMSO(180 ml) was treated gradually over several hours with cyanamide (13.0g, 0.31 mole) and K₂ CO₃ (36.8 g, 0.27 mole). The mixture was stirredfor 48 h and filtered. The filtrate was diluted with H₂ O (6 l) andglacial acetic acid (40 ml) and extracted with CH₂ Cl₂ (4×500 ml). Thecombined organic solution was washed successively with 5% NaHCO₃solution (1×500 ml) and brine (2×500 ml), dried (Na₂ SO₄), andevaporated in vacuo to dryness. The crude product was purified bychromatography on silica gel using CH₂ Cl₂ : methanol (98:2) to give 1.8g (16%) of compound 7 as a red solid, m.p. 155° C. (dec). Mass. Spec.:M⁺ =318 (q=4), 285 (q=100).

Example 7

Preparation of 1,2,4-Benzotriazine 1,4-Dioxide ##STR14##

A mixture of 1.80 g (13.73 mmole) of 8, 90% H₂ O₂ (9 ml),trifluoroacetic anhydride (13.5 ml) and Na₂ WO₄.2H₂ O (12.50 g, 38mmole) in CHCl₃ (170 ml) was stirred at room temperature for 5 days. Thereaction mixture was diluted with H₂ O (100 ml) and extracted with CHCl₃(100 ml). The organic layer was washed with H₂ O (50 ml), dried (Na₂SO₄), and the solvent removed in vacuo. The residue was chromatographedon silica gel using EtOAc-CH₂ Cl₂ (1:1) to give 0.30 g (13.4%) ofcompound 9 as a yellow solid, m.p. 204°-205° C. Anal. Calc'd. for C₇ H₅N₃ O₂ (163.13): C, 51.5; H, 3.09; N, 25.76. Found: C, 51.6; H, 3.36; N,26.01. Mass Spec. M⁺ =163 (q=100), 147 (q=50). TLC: R_(f) =0.27(EtOAc-CH₂ Cl₂, 1:1, silica gel plates). IR (nujol): 1600μ, 1460μ,1300μ, 1230μ. UV_(max) (H₂ O ): 227 (ε22,900) 252 (ε12,950): 392(ε4,080).

Example 8

Preparation of 7-Chloro-3-Hydroxy-1,2,4-Benzotriazine 1,4-Dioxide##STR15##

A mixture of 1.50 g (7.63 mmole) of 10 in 100 ml acetic acid was treatedwith 2.52 g (7.63 mmole) of Na₂ WO₄.2H₂ O and 30 ml of 30% H₂ O₂. Themixture was stirred and heated for 6 days at 50° C., then slowlyevaporated to dryness to remove H₂ O₂. The residue was boiled in 250 mlH₂ O and filtered to remove about 25 mg of starting material 12. Theaqueous solutions were then extracted with 2×250 ml portions of ethylacetate. A deep red crystalline material that was characterized as 12 byTLC and Mass. Spec. analysis formed in the partitioning mixture aboveand was collected by filtration to afford 60.0 mg of a yellowish orangesolid (3.7% yield), characterized as follows as 12, which showed goodsolubility in a mixture of hot isopropyl alcohol and water. Mass. Spec.:M⁺ =212 (q=100)(compound 10); TLC: R_(f) =0.34 (acetone, silica gelplates).

The ethyl acetate solutions above, separated from the H₂ O layer afterthe filtration to remove 12, were evaporated to dryness. The residue wasthen treated with isopropyl alcohol at room temperature to afford a dullorange solid, 0.41 g (25% yield) of 11. Mass. Spec.: M⁺ =213 (q=70);TLC: R_(f) =0.22 (acetone, silica gel plates). Compound 11 wascharacterized as the ammonium salt, C₇ H₄ ClN₃ O₃.NH₃, m.w. 230.61, asfollows. The free acid 11 was dissolved in concentrated NH₄ OH and thenchilled in ice and filtered to remove a trace of insoluble 12. The redfiltrate and washings were evaporated to dryness, leaving areddish-orange solid. The solid was treated with 50 ml of boiling1,2-dimethoxyethane, collected on a filter and washed with an additional25 ml of hot 1,2-dimethyl ether. The solid was dried over P₂ O₅ at 56°C./1.0 mm, leaving 0.244 g (87% yield) of 13 ##STR16##

Anal. Calc'd. for C₇ H₄ ClN₃ O₃ NH₃ (230.61): C, 36.5; H, 3.06; N,24.30. Found: C, 36.5; H, 3.07; N, 23.94.

UV_(max) (H₂ O): 219 (ε12,580); 265.4 (ε40,000); 4830486 (ε6,640).

Example 9

Preparation of 7-Nitro-3-Amino-1,2,4-Benzotriazine 1,4-Dioxide ##STR17##

7-Nitro-3-trifluoroacetamido-1,2,4-benzotriazine 1-oxide (15): Asolution of 7-nitro-3-amino-1,2,4-benzotriazine 1-oxide (14) (4.00 g,19.3 mmol; Parish Chemical Co.), CHCl₃ (125 ml) and trifluoroaceticanhydride (12.0 ml, 85.0 mmol) was stirred at room temperature for 44hr. The resultant light yellow solid was filtered, washed with CHCl₃ (50ml) and dried to give 5.35 g (91% yield) of the product as a yellowsolid. Anal. Calc'd. for C₉ H₄ F₃ N₅ O₄ : C, 35.7; H, 1.33; N, 23.10.Found: C, 35.7; H, 1.23; N, 23.06.

7-Nitro-3-amino-1,2,4-benzotriazine 1,4-oxide (16): To a stirredsolution of 7-nitro-3-trifluoroacetamido-1,2,4-benzotriazine 1-oxideprepared above (15) (2.50 g, 8.25 mmol) in CHCl₃ (200 ml) was added Na₂WO₄.2H₂ O (90 mg, 0.273 mmol) followed by 70% H₂ O₂ (10 ml). After 15min the solution was treated with trifluoroacetic anhydride (8.0 ml,56.7 mmol) and stirring was continued at room temperature for 64 hr. Thereaction mixture was chromatographed (EtOAc, 20% MeOH/acetone, andfinally 20% DMF/acetone) then recrystallized in acetone to give 1.20 g(65% yield) of the product (16) as an orange solid, mp 286°-288° C.(dec.). UV: λ259, 300, 345, 387, 472. Anal. Calc'd. for C₇ H₅ N₅ O₄ : C,37.70; H, 2.26; N, 31.39. Found: C, 37.70; H, 2.13; N, 30.94.

Example 10

Preparation of 3-(3-N,N-Diethylaminopropylamino)-1,2,4-Benzotriazine1,4-Dioxide ##STR18##

3-(3-N,N-diethylaminopropylamino)-1,2,4-benzotriazine 1-oxide (18): Asolution of 3-chloro-1,2,4-benzotriazine 1-oxide (17) (3.0 g, 16.5 mmol)(produced by the method of Sasse et al., U.S. Pat. No. 4,289,771) in CH₂Cl₂ (100 ml) was treated with N,N-diethylpropylenediamine (9.5 ml, 88.3mmol). After 20 hr at room temperature the mixture was diluted with1,2-dichloroethane (50 ml) and washed successively with Na₂ CO₃ and H₂O. The yellow solution was dried (Na₂ SO₄), filtered and evaporated invacuo to give 3.93 g (87% yield) of the product as a yellow solid.Recrystallization (ether/petroleum ether) yielded pure material, mp47°-48° C. Anal Calc'd. for C₁₄ H₂₁ N₅ O (18): C, 61.10; H, 7.69; N,25.44. Found: C, 61.30; H, 7.80; N, 25.61.

3-(3-N,N-diethylaminopropylamino)-1,2,4-benzotriazine 1,4-dioxide (18a):To a stirred solution of3-(3-N,N-diethylaminopropylamino)-1,2,4-benzotriazine 1-oxide 18prepared as above (1.60 g, 6.10 mmol) in CHCl₃ (50 ml) was addedtrifluoroacetic anhydride (22.0 ml). After 15 min the mixture was cooledto -10° C., 70% H₂ O₂ (10 ml) added and then stirred at room temperaturefor 20 days. The reaction mixture was dried (Na₂ SO₄), filtered andevaporated to dryness. The residue was dissolved in saturated NaHCO₃solution (50 ml) and extracted with CH₂ Cl₂ (3×150 ml). The organiclayer was dried (Na₂ SO₄), filtered and evaporated to give the product18a, 0.51 g (29% yield) as a red solid, mp 92°-94° C. NMR: δ(400 MHz,CDCl₃) 1.11 (6H, t, J=7.1 Hz, CH₃), 1.84-1.90 (2H, m, H-2'), 2.48'2.64(4H, m, NCH₂ CH₃ and H-3'), 3.68 (2H, br t, J=5.5 Hz, H-1'), 7.46 (1H,ddd, J=7.1, 7.0 and 1.2 Hz, H-6), 7.84, ddd, J=7.0, 6.9 and 1.2 Hz,H-7), 8.31 (2H, m, H-5 and 8), 8.80 (1H, br s, NH). UV: λ220, 270, 476.Anal. Calc'd. for C₁₄ H₂₁ N₅ O₂. (1/3 H₂ O): C, 56.50; H. 7.34; N,23.55. Found: C, 56.90; H, 7.15; N, 23.40.

Example 11

Preparation of7-Nitro-3-(2-N,N-Diethylaminoethylamino)-1,2,4-Benzotriazine 1,4 Dioxide##STR19##

7-nitro-3-(2-N,N-diethylaminoethylamino)-1,2,4-benzotriazine 1-oxidehydrochloride (20): A solution of 7-nitro-3-chloro-1,2,4-benzotriazine1-oxide (19) (1.60 g, 7.06 mmol) (prepared as generally shown in Sasseet al., U.S. Pat. No. 4,289,771, with (a) NaNO₂ and H₂ SO₄ at 40° C.,followed by (b) chlorination with POCl₃ at 106° C.) in CH₂ Cl₂ (50 ml)was treated with N,N-diethylethylenediamine (6.0 ml, 42.7 mmol). After16 hr at room temperature the mixture was evaporated to dryness underhigh vacuum at 60° C. The yellow solid was stirred in 20% iPrOH/ether(150 ml) for 5 hr, filtered, washed with iPrOH then petroleum ether anddried (80° C./1.0 mmHg) to give 1.80 g (74% yield) of the product 20 asyellow needle crystals. NMR δ(90 MHz, d₆ -DMSO/d₄ -MeOH) 1.25 (6H, t,J=6.0 Hz, CH₃), 3.25 (6H, m, NCH₂), 3.82 (2H, m, H-1'), 7.74 (1H, d,J=7.0 Hz, H-5), 8.52 (1H, dd, J=7.0 and 2.0 Hz, H-6), 8.91 (1H, d, J=2.0Hz, H-8).

7-nitro-3-(2-N,N-diethylaminoethylamino)-1,2,4-benzotriazine 1,4-dioxidehydrochloride (21). To a stirred solution of7-nitro-3-(2-N,N-diethylaminoethylamino)-1,2,4-benzotriazine 1-oxidehydrochloride (20; prepared as described above) (0.50 g, 1.46 mmol) inCHCl₃ (50 ml) at 0° C. was added trifluoroacetic anhydride (9.0 ml).After 30 min 70% H₂ O₂ (4.0 ml) was added and the mixture stirred atroom temperature for 3 days, then dried (Na₂ SO₄), filtered, andevaporated in vacuo to dryness to give the trifluoroacetate salt 0.67 g(45% yield). This product was dissolved in saturated NaHCO₃ solution (30ml) and extracted with CH₂ Cl₂ (3×30 ml). The dichloromethane was washedwith H₂ O, dried (Na₂ SO₄), filtered, saturated with gaseous HCl andevaporated to dryness to give 0.35 g (63% yield, 28% overall) of theproduct as a red solid, m.p. 194°-195° C. UV: λ260, 306, 388, 479. Anal.Calc'd. for C₁₃ H₁₈ N₆ O₄.HCl: C, 43.50; H, 5.34; N, 23.43. Found: C,43.20; H, 5.37; N, 23.11.

The following Examples 12-15 are directed to reductive deaminationreactions for preparing compounds of Formula (I) which are unsubstitutedat the 3-position, i.e., wherein the substituent "X" is hydrogen.

Example 12

Preparation of 1,2,4-Benzotriazine 1,4-Dioxide by Reductive Deaminationof 3-Amino-1,2,4-Benzotriazine 1,4-Dioxide ##STR20##

To a rapidly stirred solution of t-butyl nitrite (867 mg, 1.0 ml, 8.41mmol) in DMF (20 ml) at 60°-65° C. was added 3-amino-1,2,4-benzotriazine1,4-dioxide ("SR 4233") (500 mg, 2.81 mmol) (prepared by the method ofSeng et al., Angew. Chem. Internat. Edit. 11:11 (1972)) in smallportions over 5 min. Following the addition, and subsidence of theconcomitant effervescence (approx. 5 min), the solution was cooled andreduced under high vacuum to a dark waxy solid. Flash chromatography(30% EtOAc/CH₂ Cl₂) gave a yellow solid, mp 188°-189.5° C. (dec.), whichwas recrystallized from methanol to give 195 mg (43% yield) of theproduct 9 as bright yellow platelets, mp 192°-194° C. (dec.). NMR: δ(400MHz, d₆ -acetone) 8.04 (1H, ddd, J=8.5, 7, 1.5 Hz), 8.15 (1H, ddd,J=8.5, 7, 1.5 Hz), 8.42 (1H, dd, J=8.5, 1.5 Hz), 8.43 (1H, dd, J=8.5,1.5 Hz) 9.05 (1H, s, H-3). UV: λ 405, 300, 225. MS, m/z (relativeintensity) 164(9), 163(100, M⁺), 147(13), 136(19), 90(7), 78(27),76(26), 75(8), 64(9), 63(10), 52(12), 51(48), 50(28), 38(8), 37(5),30(18), 28(6), 27(7).

Anal. Calc'd. for C₇ H₅ N₃ O₂ : C, 51.54; H, 3.09; N, 25.76. Found: C,51.42; H, 3.02; N, 25.66.

Example 13

Preparation of 7-Allyloxy-1,2,4-Benzotriazine 1,4-Dioxide Via ReductiveDeamination ##STR21##

7-Allyloxy-1,2,4-benzotriazine 1,4-dioxide 24: To a stirred solution oft-butyl nitrite (271 mg, 0.312 ml, 2.63 mmol) in DMF (15 ml) at 60°-65°C. was added 7-allyloxy-3-amino-1,2,4-benzotriazine-1,4-dioxide 23 (205mg, 0.875 mmol) in small portions over 5 min. After 30 min additionalt-butyl nitrite (271 mg, 0.312 ml, 2.63 mmol) was added, and shortlythereafter the deep red solution effervesced and lightened appreciablyin color over a period of a few minutes. After an additional 30 min theresultant orange solution was reduced under vacuum to a brown solidwhich was sequentially flash chromatographed (10% EtOAc/CH₂ Cl₂) andcrystallized (CH₂ Cl₂ /petroleum ether) to give 72 mg (38% yield) of theproduct 24 as light orange crystals, mp 147°-148° C. NMR: δ(400 MHz,d6-acetone) 4.89 (2H, ddd, H-1', J_(1'),2' 5.5, J_(1'),3'cis=J_(1'),3'trans =1.5 Hz), 5.36 (1H, ddd, H-3', J_(3'),2'cis =10.5,J_(3'),3' =3, J_(3'),1' =1.5 Hz), 5.52 (1H, ddd, H-3', J_(3'),2'trans=17.5, J_(3'),3' =3, J_(3'1') =1.5 Hz), 6.14 (1H, ddt, H-2',J_(2'),3'cis =10.5, J_(2'),1' =5.5 Hz), 7.70 (1H, d, H-8, J₈,6 =2.5 Hz),7.74 (1H, dd, H-6, J₆,5 =9.5, J₆,8=2.5 Hz), 8.33 (1H, d, H-5, J₅,6 =9.5Hz), 8.93 (1H, s, H-3). UV: λ425, 410, 365, 355, 320, 245, 200. MSm/z/(relative intensity) 220(4), 219(34,M⁺), 103(4), 77(4), 75(4),63(13), 62(4), 42(3), 41(100), 39(16).

Anal. Calc'd. for C₁₀ H₉ N₃ O₃ : C, 54.79; H, 4.14; N, 19.17. Found: C,54.73; H, 4.16; N. 19.15.

Example 14

Preparation of7-(3-N-Ethylacetamido-2-acetoxypropoxy)-1,2,4-Benzotriazine 1,4-DioxideVia Reductive Amination ##STR22##

To a stirred solution of t-butyl nitrite (185 mg, 1.79 mmol) in DMF (5ml) at 60° C. was added via syringe a solution of7-(3-N-ethylacetamido-2-acetoxypropoxy)-3-amino-1,2,4-benzotriazine1,4-dioxide (25) (125 mg, 0.329 mmol) in DMF (5 ml) over a period of 1min. After 5 min additional t-butyl nitrite (217 mg, 2.10 mmol) wasadded and an immediate reaction occurred, as evidenced by the evolutionof a gas and a change in color of the solution from red to light orange.After an additional 10 min the solution was stripped to a yellow/brownsolid and eluted through silica gel with 5% MeOH/CH₂ Cl₂ to give 119 mgof a yellow oil. Recrystallization from CH₂ Cl₂ /ligroin gave 90 mgyellow solid (75% yield), mp 179°-180.5° C. NMR: δ(400 MHz, d₄-methanol, mixture of rotamers, ratio approx. 2:1) 1.12, 1.22 (t's, 1:2,3 H total, J=7 Hz), 2.06, 2.07 (s's, 2:1, 3 H total), 2.11, 2.17 (s's,2:1, 3 H total), 3.41-3.92 (m, 4 H), 4.34-4.48 (m, 2 H), 5.48-5.58 (m, 1H), 7.76-7.86 (m, 2 H), 8.36-8.42 (m, 1 H), 9.04, 9.06 (s's, 2:1, 1 Htotal). UV: λ420, 405, 365, 350, 315, 240, 200. MS: m/z (relativeintensity) 365(0.5), 364(1.4, M⁺), 349(0.5), 348(1.1), 347(0.5),332(1.2), 331(3.6), 187(7), 186(66), 102(6), 100(21), 84(30), 63(6),58(100), 56(8), 43(65), 42(9), 41(5), 30(14), 29(5), 28(8).

Example 15

Preparation of 7-Nitro-1,2,4-Benzotriazine 1,4-Dioxide via ReductiveDeamination ##STR23##

To a stirred solution of t-butyl nitrite (88 mg, 0.85 mmol) in DMF (5ml) at 60° C. was added 7-nitro-3-amino-1,2,4-benzotriazine 1,4-dioxide(14) (38 mg, 0.17 mmol). After 30 min the addition of further t-butylnitrite (175 mg, 1.70 mmol) to the dark red slurry was immediatelyfollowed by a change in coloration and effervescence. After anadditional 10 min the orange solution was reduced to a red solid invacuo and chromatographed with 1% AcOH/CH₂ Cl₂ to give 3 mg of theproduct 27 as a yellow solid (10% yield). NMR δ(90 MHz, d₆ -dimethylsulfoxide) 7.68 (d, 1H, J=9.2 Hz), 7.92 (dd, 1H, J=9.2, 2.2 Hz), 8.10(d, 1H, J=2.2 Hz), 8.65 (s, 1H)). UV: λ420, 310, 240, 205.

MS:m/z (relative intensity) 209 (9), 208 (100, M⁺), 192 (54), 181 (14),162 (16), 105 (9), 77 (28), 75 (52), 74 (27), 63 (21), 62 (16), 30 (77),18 (26).

Example 16

In Vivo Assay for Activity in Combination with Radiation

Compounds of the invention were tested in vivo for activity by the assayof Brown, J. M., Radiation Res (1975) 64:633-47, incorporated herein byreference. For this assay, SCCVII carcinomas in female C3H mice weighing20-25 g were used. These mice were bred under specific pathogen-freeconditions and were 3-4 months old at the beginning of each experiment.The SCCVII tumor was grown intradermally in the flank from aninoculation of 2×10⁵ tumor cells taken from the 2nd-8th in vitro passageof the tumor cells after removal from the previous in vivo tumor. Twotumors per mouse were implanted, and were used as subject tumors whenthey reached a volume of approximately 100 ml. At this point the tumorscontained approximately 20% hypoxic cells.

The test compound was tested at a fixed injected dose of either 5mmol/kg or 2/3 of the LD₅₀ (whichever was lower). Suitable controls oftest compound-injected but nonirradiated and saline-injected andirradiated mice were also included. A fixed radiation dose of 20 Gy wasapplied at variable intervals of 2 hr after to 3 hr before injection ofthe drug. By using these intervals, the results give an indication ofboth the optimum irradiation time and the extent of extra cell killingcompared to radiation alone. The results of such time-course experimentsusing 3-amino-1,2,4-benzotriazine 1,4-dioxide are shown in FIG. 2. Theyshow enhanced cell killing compared to radiation only, more than wouldhave been expected on the basis of additivity of the two individualcytotoxicities. The similar increased cytotoxicity when the drug isgiven before or after radiation indicates selective toxicity to thehypoxic cells rather than a radiosensitizing effect of the benzotriazinedioxide.

Irradiation of the SCCVII tumors was done by irradiatingnonanaesthetized tumor-bearing mice in a Plexiglas box. Irradiationconditions were 250 kVp X-rays, 15 mA, FSC 33 cm, added filtration of0.35 mm Cu, half value layer 1.3 mm Cu, and a dose rate of 317 rad/min.

The amount of cell killing was judged by survival rate of dissected andcultured tumor cells as follows. The tumor-bearing mice were killed 24hr after irradiation, and tumors were dissected from the skin, cut intoseveral pieces, and made into a fine brei by high-speed chopping with arazor blade attached to a jigsaw. The brei was added to 30 ml of Hank'sbuffered salt solution (HBSS) containing 0.02% DNase, 0.05% promase, and0.02% collagenase. The suspension was stirred for 30 min at 37° C.,filtered, and centrifuged at 1,600 rmp for 10 min at 4° C. The cellpellet was resuspended in complete Waymouth's medium plus 15% fetal calfserum (FCS) and an aliquot mixed with trypan blue and counted with theuse of a hemacytometer. Suitable dilutions of this serum plated into 60-or 100-mm polystyrene petri dishes (Lux Scientific Corp) in 5 or 15 mlof medium. After incubation for 13 days, the colonies were fixed andstained, and those containing 50 cells or more were counted. Thedilution yielding an average count of 25-100 colonies in a 60 mm dishwas used in calculation of results.

Example 17

Cytotoxicity Tests

Cytotoxicity tests were carried out using 3-amino-1,2,4-benzotriazine1,4-dioxide and a variety of aerobic and hypoxic cells in culture(human, mouse, and hamster). The cells in spinner flasks were gassed forone hour at 37° C. with either air or nitrogen containing 5% CO₂ priorto adding the specified amounts of the drug. FIGS. 1A, 1B and 1C showthe results for cell survival of mouse, hamster and human cells atvarious concentrations of 3-amino-1,2,4-benzotriazine 1,4-dioxide. Itwas found that only 1 to 2% of the drug concentration under aerobicconditions was required to get equal cell killing under hypoxia. Thisratio of selective hypoxic toxicity (50-100) is higher than that for anycompound so far reported in the literature.

Example 18

Determination of LD₅₀

LD₅₀ is determined in BALB/c female mice (weighing 20-25 g) followingintraperitoneal (ip) injection, unless the compound tested has lowlipophilicity and is very soluble, wherein intravenous (iv)administration is used. LD₅₀ values at 1, 2, 5, and 60 days aredetermined by administering graded doses of the drug dissolved inphysiological saline immediately prior to injection.

Example 19

Radiosensitivity in Vitro

The results of assays to determine the concentration of drug necessaryto produce a sensitizer enhancement ratio of 1.6 of hypoxic cells inculture are as follows:

    ______________________________________                                        Compound               C.sub.1.6  (mM)                                        ______________________________________                                        7-chloro-3-amino-1,2,4-benzotriazine                                                                 3.3                                                    1-oxide                                                                       6(7)-methoxy-3-amino-1,2,4-benzotriazine                                                             ˜1.0                                             1,4-dioxide                                                                   3-hydroxy-1,2,4-benzotriazine 1,4-dioxide                                                            ˜2.0                                             ______________________________________                                    

Modifications of the above described modes for carrying out theinvention that are apparent to those of skill in the chemical,pharmaceutical, medical, and related arts are intended to be within thescope of the following claims.

Example 20

Enhanced Tumor Cell Toxicity Using Hydralazine

Hydralazine is an antihypertensive drug which acts by relaxing thesmooth muscle around blood vessels. This has the effect ofpreferentially shunting blood flow into normal tissues and away fromtumors, which process produces immediate hypoxia in the tumors. If3-amino-1,2,4-benzotriazine 1,4-dioxide is given in conjunction withthis agent, there is a massive increase in tumor cell killing. In thisexperiment, neither hydralazine nor the aforementioned benzotriazinecompound produced any significant cell killing in the SCCVII tumor,whereas the combination of the two reduced survival by a factor of 10³(i.e., only 1 cell in every 1000 was left viable). The experimentalprocedures are the same as described in Example 9, and the results areshown in FIG. 3.

Example 21

Physicochemical and Biological Properties of Some 1,2,4-Benzotriazine1,4-Dioxides

The following table sets forth various properties of compounds 22, 14,18a, and 21 as determined by the inventors herein:

    ______________________________________                                              Mol.    Solubility                                                                             Log  E1/2.sup.b                                        Cpd.  Wt.     (mM)     p.sup.a                                                                            (mV)  RHT.sup.c                                                                          HCR.sup.d                                                                           LD.sub.50.sup.e                  ______________________________________                                        22    178.2   13.50    -0.32                                                                              -332  1    100   0.50                             14    223.2   2.00     -0.97                                                                              -133  2    140   1.00                             18a   291.4   >181     -1.34                                                                              -348  3    100   0.25                             21    358.9   96.40    -0.20                                                                              -140  3    211   0.40                             ______________________________________                                         .sup.a Log of the octanolwater partition coefficient as measured by the       method of Fujita et al., J. Amer. Chem. Soc. 86:5175 (1964), using pH 7.4     buffer.                                                                       .sup.b Polarographic halfwave reduction potentials measured in Britton &      Robinson pH 7.4 buffer using a dropping mercury electrode.                    .sup.c Relative Hypoxic Cytoxicity: Ratio of equitoxic concentrations of      22:analog for HA1 cells attached, under hypoxic conditions. Exponentially     growing cells were placed in suspension culture and gassed for 90 min in      nitrogen or air prior to addition of drugs. Samples were removed              periodically for a survival determination, and the ratios determined from     a comparison of the resulting survival curves.                                .sup.d Hypoxic Cytoxicity Ratio: Ratio of equitoxic concentrations of eac     analog for HA1 cells attained under hypoxic:aerobic conditions. Treatment     conditions as above.                                                          .sup.e Balb/c female mice 3-5 months of age were used in the LD.sub.50        experiments. LD.sub.50 was evaluated as described in Example 18.         

As may be readily deduced from the table, novel compounds 14, 18a,and 21exhibit significantly enhanced cytotoxicity against hypoxic HA-1 tumorcells in vitro compared to 3-amino-1,2,4-benzotriazine 1,4-dioxide (22),while retaining the high differential cytotoxicity against hypoxic cellscompared to aerobic cells. These results suggest that these drugs willbe more tumor-specific and therefore more effective as antitumor agentsin vivo.

Example 22

Fractionated Radiotherapy Using 3-Amino-1,2,4-Benzotriazine 1,4-Dioxide

The following experimental work establishes that pre- orpost-irradiation treatment of cells in vitro with compound 22 underhypoxic conditions radiosensitizes the cells even when drug is notpresent during radiation exposure and the cells are aerobic.

a.) FIG. 4 shows the results of experiments in which the survival ofChinese hamster ovary (CHO) cells after graded doses of x-rays wasdetermined either with a hypoxic compound 22 exposure given before orafter irradiation. The preirradiation treatments consisted of 20 μM drugfor exposure times of 1.0 (□ in FIG. 4), 1.5 (Δ), 2.0 (⋄) and 2.5 () hduration prior to cells being reaerated and irradiated. These drugtreatments alone reduced cell survival to approximately 32, 19, 8 and 2%respectively. The postirradiation drug treatment consisted of 20 μM for1.5 h (+), which alone reduced cell survival to 23%. Compared to thesurvival curve for cells exposed to pre- or post-irradiation hypoxiaonly (▪), treatment with compound 22 sensitized cells to aerobicirradiation. The sensitization was predominantly a change in the slopeof the radiation survival curve. D_(o) decreased from 1.34 Gy (95%confidence limits: 1.09-1.76 Gy) to 0.80 Gy (95% confidence limits:0.73-0.88 Gy), based on least-squares regression analysis of pooled datafor the exponential portions of the survival curves for control andtreated cells. However, sensitization at low radiation doses was alsoreadily apparent. Survival ratios for "no drug: drug-treated" CHO cellsirradiated with doses of 1-3 Gy averaged a factor of 3.6. The amount ofthe radiosensitization produced did not vary with the severity of thedrug treatment.

b.) The data of section (a.) on radiosensitization of aerobic cells atlow radiation doses by hypoxic activation led us to test the feasibilityof obtaining preferential radiosensitization of tumors in vivo. Ourprotocol was to use eight doses of 2.5 Gy/dose in four days (irradiating2×/day). Because of the in vitro data showing that radiosensitizationcould only be achieved by hypoxia activation, either before or afterirradiation, and because we did not want to make the tumors hypoxicbefore irradiation (as this would make them resistant), we made thetumors hypoxic after irradiation using the vasoactive drug hydralazine(HDZ) at the same time as injecting compound 22. We used two differenttypes of controls. First, compound 22 alone before each dose ofradiation, and second, the potent hypoxic cell sensitizer SR 2508 (a2-nitroimidazole, DuPont, undergoing Phase III clinical trials inEurope) before each dose. The effectiveness, or lack of effectiveness,or SR 2508 indicates whether the radiation response of the tumor isbeing governed by hypoxic, or aerobic, cells respectively. We assayedthe efficacies of the treatments using clonogenic cell survival and alsoregrowth delay. FIGS. 5 and 6 show the results.

As FIG. 5 illustrates, there was no effect of SR 2508 (1000 mg/kg) givenbefore each radiation dose, but compound 22 (0.08 mmole/kg) given aloneor with hydralazine (HDZ) produced a large enhancement of the radiationresponse. Part of this is attributable to an additive response(crosses), but the additional cell killing is the result ofradiosensitization of aerobic cells.

In FIG. 6, it may be seen that SR 2508 (1000 mg/kg) before eachradiation dose had no radiosensitizing effect, but compound 22 (0.08mmole/kg) alone before irradiation or with HDZ after irradiationproduced a large increase in effect compared to radiation or drug alone.

The major and unexpected result of these experiments is theradiosensitization of the tumors by compound 22 given before eachradiation dose without the addition of hydralazine. This cannot beaccounted for by a radiosensitization of hypoxic cells, since SR 2508 (ahypoxic radiosensitizer) is ineffective. Thus, it is aradiosensitization of aerobic tumor cells.

This would not be useful if it radiosensitized aerobic normal cells. Wetested this by performing the same eight fraction protocol on theresponse of normal mouse skin using a skin reaction scoring scalepreviously used by us. FIG. 7 shows the result. There is noradiosensitization of normal skin.

In conclusion, the data show that the aerobic cells of tumors can beradiosensitized in multifraction regimes similar to those used inradiotherapy. The radiosensitization is tumor specific (i.e., does notoccur in normal cells), and appears to be the result of activation byhypoxic areas in the tumors.

We claim:
 1. A method of selectively killing hypoxic tumor cellssensitive to the formula in a host comprising administering to said hostan effective amount of a pharmaceutical composition comprising acompound of the formula ##STR24## wherein X is NH₂ ; NHR or NRR; whereineach R is independently an alkyl of 1-4 carbon atoms or acyl of 1-4carbon atoms, or wherein in the case of NRR the two R groups may belinked together to form a morpholino, pyrrolidino or piperidino ring,and wherein R may be further substituted with OH,. NH₂, alkyl (1-4C)secondary amino, dialkyl (1-4C) tertiary amino, morpholino, pyrrolidino,piperidino, alkoxy (1-4C), or halogen substituents;n is 1; and Y¹ and Y²are independently either H; nitro, halogen; hydrocarbyl (1-14C)including cyclic and unsaturated hydrocarbyl, optionally substitutedwith 1 or 2 substituents selected from the group consisting of halogen,hydroxy, epoxy, alkoxy (1-4C), alkylthio (1-4C), primary amino (NH₂),lower alkyl (1-4C) secondary amino, dialkyl (1-4C) tertiary amino,dialkyl (1-4C) tertiary amino where the two alkyls are linked togetherto produce a morpholino, pyrrolidino or piperidino, acyloxy (1-4C),acylamido (1-4C) and thio analogs thereof, acetylaminoalkyl (1-4C),carboxy, alkoxycarbonyl (1-4C), carbamyl, alkylcarbamyl (1-4C),alkylsulfonyl (1-4C) or alkylphosphonyl (1-4C), wherein the hydrocarbylcan optionally be interrupted by a single ether (--O--) linkage; orwherein Y¹ and Y² are independently either morpholino, pyrrolidino,piperidino, NH₂, NHR', NR'R' O(CO)R', NH(CO)R', O(SO)R', or O(POR')R' inwhich R' is a hydrocarbyl (1-4C) which may be substituted with OH, NH₂,alkyl-(1-4C) secondary amino, dialkyl (1-4C) tertiary amino, morpholino,pyrrolidino, piperidino, alkoxy (1-4C), or halogen substituents, or apharmacologically acceptable salt of said compound.
 2. The method ofclaim 1, wherein X is NH₂.
 3. The method of claim 2, wherein Y¹ and Y₂are both H.
 4. The method of claim 2, wherein Y¹ is H and Y² is nitro.5. The method of claim 1, wherein X is --NH--CH₂ --(CH₂)m--CH₂ --NR₁ R₂wherein m is an integer in the range of 0-4 inclusive, and R₁ and R₂ areindependently selected from hydrogen or lower alkyls or together form apiperidino or pyrrolidino ring.
 6. The method of claim 5, wherein m is 1or 2 and Y¹ and Y² are independently selected from the group consistingof H and nitro.
 7. The method of claim 1, wherein said compound is3-amino-1,2,4-benzotriazine 1,4-dioxide.